PROJECT SUMMARY The receptor-ligand complex of scavenger receptor class B type I (SR-BI) and high density lipoprotein (HDL) is responsible for removal of cholesteryl ester (CE) from the body. This process is crucial to the prevention of hypercholesterolemia and atherosclerosis. Recently, a study in mice reported that knocking out (KO) procollagen endopeptidase enhancer 2 (PCPE2), an extracellular matrix protein, resulted in a paradoxical finding of increased plasma HDL associated with a greater extent of atherosclerosis. Additional investigation demonstrated that in spite of increased SR-BI levels in livers of PCPE2 KO mice, both plasma cholesterol clearance and sterol excretion were reduced. Based on these novel findings, we suggest that PCPE2 either directly interacts with HDL particles or with SR-BI, or a combination of the two, to stabilize HDL interaction(s) with the receptor and/or provide a scaffold to hold SR-BI in place within the membrane. In this multi-PI application Dr. Sorci-Thomas and Dr. Sahoo propose to carry out collaborative studies to test the hypothesis that PCPE2 functionalizes SR-BI receptors on the cell surface to facilitate bidirectional cholesterol transport. In Aim 1 we investigate how SR-BI works in synergy with PCPE2 to maintain cellular cholesterol homeostasis. A newly-generated CRISPR/Cas9 PCPE2-deleted 3T3 cell line and a new tissue-specific PCPE2 knockout mouse model will be used to elucidate the physiological relevance of the partnership between SR-BI and PCPE2, and define how they work together to promote receptor translocation and bidirectional cholesterol transport. Preliminary data already support a unique physiological role for this partnership in mouse adipose tissue, in isolated mouse adipocytes and in differentiated 3T3 cells, suggesting that these two proteins play a significant role in lipid storage. Aim 2 will explore the mechanism defining how SR-BI partners with PCPE2 to enhance CE uptake from HDL. Specifically, we will determine if PCPE2 facilitates SR-BI oligomerization on the plasma membrane, a process that is postulated to enable HDL-CE delivery into cells, perhaps by orchestrating interactions between HDL and SR-BI. This will be tested in both freshly isolated adipocytes as well as in our PCPE2-deleted 3T3 cell line recently created using CRISPR/Cas9 methods. In Aim 2, utilizing a combination of mutagenesis, and innovative biochemical and mass spectrometry strategies, we will identify specific regions of interaction between PCPE2 and SR-BI or PCPE2 and HDL. Together, these innovative studies will exploit the complementary expertise of the Sorci-Thomas and Sahoo labs to identify novel mechanisms that regulate SR- BI's function in bidirectional cholesterol transport. The findings from these studies will help identify new therapeutic strategies for preventing hypercholesterolemia and its associated pathologies such as atherosclerosis.